'Tandem' Primers for PCR Mutagenesis

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Apr 7 13:21:20 EST 1994

> Howdy All,
>      I was curious if you could use two sets of primers to produce site-
> directed mutants.  The first set are the 'universal' primers that 'cover' 
> the entire length-plus of the gene of interest.  The second set of primers 
> are somewhere within the gene and bear the desired point mutation; these 
> primers would necessarily be complementary to one another.  The 'figure' 
> below illustrates my question:
> Universal,  ________\         ____*__\                  & Mutagenic primers
>            <----------------------------------------    Template DNA +
>             ||||||||||||||||||||||||||||||||||||||||    Base pairs
>            ---------------------------------------->    Template DNA -
> Mutagenic,                   \----*---      \-------    & Universal primers
>                       *********************             Coding region 
> Now run PCR (in presence of dNTP's etc.).  Will Taq 'respect' both primers 
> per strand and amplify a site-directed mutant?  Will significant problems 
> arise from dimerization of the mutant oligos?  Will Taq make a 
> phosphodiester bond when the two growing segments meet?  Any other thoughts 
> or comments would be most appreciated.  Cheers,  Shaun
>   =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 
>   = Shaun D. Black, PhD     | Internet:     shaun at jason.uthct.edu         = 
>   = Dept. of Biochemistry   | University of Texas Health Center, at Tyler = 
>   =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 
Dear Shaun:
	I don't think this strategy is going to work.  The first reason is
that Taq does not make phospodiester bonds between two segments of DNA but
it does so only between the 3' end of a growing chain and the 5' end of the
dNTP.  I think the phosphodiester bond you mention may be probably Ligase's
	In fact, your description reminded me of another paper (Efremov
et al Acta Hematol. 85:66 Mutant oligonucleotide extension amplification:
a nonlabelling PCR-based assay for the detection of point mutations.) used
for detecting the presence of point mutations.
The only difference was their primer had the mutation at the 3' end of the
primer.  So, an amplification will result in product from the internal mutant
primer.  In wild types, the full length product is
formed but in mutant, the binding of the mutant primer did 2 things.
Its binding "interfered" with polymerization from the external consensus 
primer and secondly if the sizes are appreciably different, it will compete
with the external primer (I suppose more successfully) and result in fragments
that are solely the result of one external and one internal mutant primer.
Unless you play with primer concentrations extensively, the only product 
you may get are these two products rather than the one full length.
	Best regards,
Raj Shankarappa
bsh at med.pitt.edu

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